The Human Umbilical Cord Mesenchymal Stem Cell Derived Exosomes Accelerate The Repair Of Large-scale Bone Defects Via Promoting Angiogenesis

BACKGROUNDThe repair of large-scale bone defects caused by trauma,tumor and deformity is always a great challenge for clinicians.The emergence and rapid development of bone tissue engineering has opened a new way for the repair of large-scale bone defects.However,the clinical effect of various bone grafts for the repair of bone defects is not satisfactory,and the key factor is that the poor ability of angiogenesis.In vitro and vivo experiments,it has been found that stem cells combined with biomaterials can effectively improve the ability of angiogenesis.However,there are still some problems in this kind of treatment,such as limited cell sources,low efficiency of induced proliferation,immune rejection,potential tumorigenicity,ethical disputes,and so on.In recent years,with the development of research on stem cells promoting tissue repair,it is found that the exosomes which produced by stem cells may play an important role in it,and it is expected to bring new hope for the treatment of large bone defects.OBJECTIVEIn the present study,we isolated and identified exosomes from human umbilical cord mesenchymal stem cells(^uMSCEXOs).Meanwhile,the hyaluronic acid(HA)hydrogel(Gel)delivery system was constructed by microfluidics,and the exosomes was encapsulated in it to achieve effective and long-term release of exosomes.To verify the role of ^uMSCEXOs in promoting bone repair by accelerating angiogenesis through fracture and critical bone defect models in vivo.To further verify the effect of ^uMSCEXOs on vascular endothelial progenitor cells'angiogenic differentiation through cell experiment in vitro.Finally,through molecular biology,bioinformatics,and other methods,we screened out the key substances in ^uMSCEXOs,and explored the internal molecular mechanism of promoting angiogenesis.This study will provide a new theoretical basis and research direction for the application of exosomes in the treatment of large bone defects in the future.MATERIALS&METHODS1.Preparation and characterization of ^uMSCEXOs and the EXOs/Gel composites(1)The exosomes were isolated from the uMSCs and HEK293 cells by ultracentrifugation.Then,these exosomes were observed and identified through transmission electron microscopy,nanoparticle tracking analysis and western blotting.(2)HA hydrogel microspheres were synthesized by microfluidics technology,and the exosomes were encapsulate in it.The loading and sustain-release capability of the hydrogel microspheres were detected by simulated body fluid immersion and nanoparticle tracking analysis.2.In vivo animal experiments(1)We established the animal model of fracture of femoral shaft in rats.Then we injected different materials(^uMSCEXOs/Gel,^HEK293EXOs/Gel and Gel)into the fracture sites,the fracture healing and the angiogenesis in callus were evaluated by X-ray,Micro-CT and histological analysis.(2)We established the animal model of critical-sized calvarial defects in rats.And we prepared polycaprolactone/mesoporous bioactive glass scaffolds through 3D printing technology(nano HA/PCL scaffolds,n HP).Then,according to the grouping(^uMSCEXOs/Gel/n HP,^HEK293EXOs/Gel/n HP,Gel/n HP and Blank),different components were implanted into the bone defect area.After operation,Micro-CT and histological analysis were performed to evaluate the repair of bone defects and the change of tissue in the bone defect area.(3)To clarify the role of ^uMSCEXOs in bone repair in this study through the above animal experiments in vivo.3.The experiment of ^uMSCEXOs in vitroGroups:^uMSCEXOs/Gel,^HEK293EXOs/Gel,Gel,blank.Endothelial progenitor cells were selected as the research object in this part of experiment in vitro.Exosomes were co-cultured with endothelial progenitor cells to observe internalization of ^uMSCEXOs by encapsulated EPCs.And the CCK-8 analysis was used to detect the effects of each group on cell proliferation.Transwell and scratch assay were used to evaluate the effects of each group on cell migration.Tubule formation assay was used to evaluate the effects of each group on cell angiogenesis.The expression of angiogenesis related genes in each group were detected through the RT-PCR technology.Finally,to confirm the role of ^uMSCEXOs in EPCs angiogenesis.4.Mechanism of ^uMSCEXOs promoting angiogenesisFirstly,the content of mi RNA in ^uMSCEXOs and ^HEK293EXOs was determined by high-throughput sequencing technology,then compared with the content of mi RNA of uMSCs which was published on the Internet.The highest content of the mi RNA was selected as the next research object.Then,we constructed ^uMSCEXOs with different mi RNA-21 expression levels,and these exosomes were observed and identified through transmission electron microscopy and western blotting.These exosomes were co-cultured with endothelial progenitor cells,and the effect on the ability of angiogenesis of endothelial progenitor cells was evaluated through the tubule formation assay in vitro.The effect of different mi RNA-21 levels of ^uMSCEXOs on the angiogenesis in vivo was evaluated through the Chick Chorioallantoic Membrane assay.Finally,the expression of angiogenesis related genes and proteins in endothelial progenitor cells was detected by RT-PCR technology,and we speculate on the underlying molecular mechanism.RESULTS1.Preparation and characterization of EXOs and the EXOs/Gel compositesIn this part of the experiment,the exosomes were separated and extracted from uMSCs and HEK293 cells by ultracentrifugation.The size of the exosomes was about60-120 mm and the concentration of the exosomes was 4.2×10⁵ particles/ml.The results of western blotting showed that CD81,CD63 and CD9 were all positive.The above experimental results showed that we have successfully isolated and extracted exosomes.The results of MRI showed that methacrylic anhydride modified HA was successful.The HA-Gel sustained-release system could be injected into different shapes of models to be solidified and molded into various shapes after UV irradiation.The result of simulated body fluid immersion experiment showed that after the HA-Gel was soaked in the simulated body fluid,the ^uMSCEXOs was released slowly with the degradation of hydrogel.After immersed for 20 Days,there was still have 40%of ^uMSCEXOs remains in HA-Gel.2.In vivo animal experimentsWe first established a rat femoral shaft fracture model.According to the experimental group,the rats were injected with different components of hydrogel materials at the fracture site.All the wounds of animals were healed well.After 2-4 weeks of operation,the width of callus in group ^uMSCEXOs/Gel was significantly larger than in the control groups.Micro-CT scan at the fracture at 2 weeks after operation showed that the bone mineral density and bone volume of the callus in the ^uMSCEXOs/Gel group were higher than those in the control groups.The results of angiography showed that the number and volume of neovascularization in the ^uMSCEXOs/Gel group were significantly higher than those in the control groups.The results of histological analysis showed that the expression of CD31 in the ^uMSCEXOs/Gel group was also significantly higher than that in the control groups.Next,we successfully established the bilateral critical-sized calvarial defect model in the rats.According to the experimental group,different composite materials were implanted into the bone defect area.The results of micro-CT in 4 and 8 weeks after operation showed that the bone mineral density,the volume of new bone,the density and thickness of trabecula in ^uMSCEXOs/Gel scaffolds group were significantly higher than those in the control groups.The results of immunohistochemistry further showed that the expression of osteogenic and angiogenic related proteins in the internal tissue of the material in the ^uMSCEXOs/Gel group was significantly stronger than all the control groups.The results of in vivo experiments showed that with the addition of ^uMSCEXOs,the repair effect of fracture and bone defect was obviously enhanced.3.The experiment of ^uMSCEXOs in vitro.In this part of the experiment,after stained and co-cultured with endothelial progenitor cells for 24 hours,the ^uMSCEXOs can be seen entered endothelial progenitor cells(EPCs)successfully under fluorescence microscope.Then,according to the experimental group,different composites were co-cultured with EPCs.The results of cell proliferation showed that compared with the control group,the ^uMSCEXOs significantly promoted the proliferation of endothelial progenitor cells.The results of Transwell and scratch assay showed that ^uMSCEXOs significantly enhanced the ability of migration of EPCs.The results of tubule formation assay showed that ^uMSCEXOs significantly improved the ability of EPCs to form tubes.The final PCR results showed that the expression of vascular related genes(VEGFA,CD31,HIF-1?)in EPCs was significantly increased after the intervention of ^uMSCEXOs,but there was no significant difference between the control groups.The results showed that the proliferation,migration and vascularization of EPCs were significantly enhanced by the addition of ^uMSCEXOs.4.Mechanism of ^uMSCEXOs promoting angiogenesisIn this part of the experiment,the results of high-throughput sequencing showed that the mi RNAs contained in ^uMSCEXOs,^HEK293EXOs and uMSCs were highly specific.And only the mi RNA-21 was the highest express in both ^uMSCEXOs and uMSCs.Combined with the literature reports,we speculated that mi RNA-21 might be the key factor for the angiogenesis in ^uMSCEXOs.Then,we constructed and identified ^uMSCEXOs with different mi RNA-21 expression levels.The results showed that when the expression of mi RNA-21in ^uMSCEXOs was inhibited in the experiments of tube formation,chick embryo chorioallantoic membrane and RT-PCR,the tube formation ability of EPCs(the length and number of neovascularization in tube formation experiment and chick embryo chorioallantoic membrane experiment)and the expression of related angiogenic genes and proteins(VEGFA,HIF-1?)were significantly decreased,while NOTCH1 and DLL4 was decreased significantly.These results suggest that exosomal mi R-21 contributed to the ^uMSCEXO-mediated angiogenic function of EPCs and promoted angiogenesis by inhibiting the NOTCH/DLL4 pathway and inducing VEGFA and HIF-1?expression.CONCLUSIONS1.We separated and extracted the exosomes from uMSCs by ultracentrifugation technology,and identified them through transmission electron microscopy,nanoparticle tracking analysis and western blotting,which verified the correctness of ^uMSCEXOs,indicating that the ultracentrifugation is a safe,reliable and efficient method for exosomes extraction.2.The HA-Gel microspheres and HA-Gel sustained release system prepared by microfluidics technology.After immersed in simulated body fluid for 20 days,there was still more than 40%exosomes remain in the hydrogel sustained release system.It indicates that the HA-Gel sustained release system can achieve effective and long-term release for exosomes.3.We successfully established a rat femoral shaft fracture model and a critical bone defect animal model in rats.Then,we injected different exos/hydrogel composites into the bone defect site.After the operation,we compared the effects of bone repair,angiogenesis and the composition of the graft area by imaging and histology analysis.The results showed that the effect of bone repair and angiogenesis in the ^uMSCEXOs/Gel group were significantly better than those in the control groups,and the expression of angiogenesis related proteins in the operation area in the ^uMSCEXOs/Gel group was significantly higher than that in the other groups.The results showed that ^uMSCEXOs/Gel can effectively promote the progress of bone repair and enhance the effect of angiogenesis in vivo.4.Through cell experiments in vitro(cell proliferation,cell migration,tubule formation,etc.),it is verified that ^uMSCEXOs can effectively promote the proliferation,migration and vascular differentiation of EPCs.5.Through high-throughput sequencing,we identified mi RNA-21 as the most abundant micro RNA in ^uMSCEXOs.In addition,we further verified the important effect of mi RNA-21 in the vascular differentiation of endothelial progenitor cells promoted by ^uMSCEXOs.And we also speculated that mi RNA-21 may play a role by regulating NOTCH1/DLL4/VEGFA signaling pathway.